Structural explanation for the role of Mn2+ in the activity of phi6 RNA-dependent RNA polymerase.
The biological role of manganese (Mn(2+)) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage 6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn(2+) by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn(2+)-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn(2+), reduced RNA binding and a compromised elongation rate. Loss of Mn(2+) binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn(2+) coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn(2+) is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.
Poranen
Minna M
MM
Institute of Biotechnology and Department of Biological and Environmental Sciences, Viikki Biocenter, P.O. Box 56 (Viikinkaari 5) 00014 University of Helsinki, Finland.
Salgado
Paula S
PS
Koivunen
Minni R L
MR
Wright
Sam
S
Bamford
Dennis H
DH
Stuart
David I
DI
Grimes
Jonathan M
JM
D017126:Bacteriophage phi 6 / Q000201:enzymology*; D001665:Binding Sites; D006160:Guanosine Triphosphate / Q000737:chemistry; D008345:Manganese / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D012313:RNA / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D014764:Viral Proteins; D008345:Manganese; D012313:RNA; D006160:Guanosine Triphosphate; D012324:RNA-Dependent RNA Polymerase